A large amount of crop loss and plant damage is incurred each year due to plant diseases caused by two classes of fungi: Ascomycetes, causing a large number of leaf spots, blights, soil-born and post-harvest diseases; and Basidiomycetes, causing rust, smuts, bunts and soil born-diseases. Also, Oomycetes cause a number of plant diseases including downy mildews, leaf blights and soil-born diseases.
Zymoseptoria tritici, also known as Septoria tritici, also known as Mycosphaerella graminicola, also known as SEPTTR, is an ascomycete in the family Mycosphaerellaceae. This fungus, a species of filamentous fungus, is a wheat plant pathogen that causes Septoria leaf blotch. Septoria leaf blotch is difficult to control due to the development of resistance to multiple fungicides.
Zymoseptoria tritici infects its host through the stomata. There is a long latent period of up to two weeks following infection before symptoms develop (Orton, E. S. et. al., (2011) Mycosphaerella graminicola: from genomics to disease control. Molecular Plant Pathology 12(5):413-424). The fungus evades host defenses during the latent phase, followed by a rapid switch to necrotrophy immediately prior to symptom expression 12-20 days after penetration.
Wheat yields can be reduced by 30-50% due to losses caused by Septoria leaf blotch (STB) with a huge economic impact (Eyal, Z. et. al., (1987) The Septoria Diseases of Wheat: Concepts and Methods of Disease Management. Mexico, DF: CIMMYT). Global costs for fungicides to manage STB total hundreds of millions of dollars each year (Hardwick, N. V. et. al., (2001) Factors affecting diseases of winter wheat in England and Wales, 1989-98. Plant Pathol 50: 453-462; McDougall, P. (2006) Phillips McDougall Agriservice Report. Scotland, UK: Pathhead, Midlothian).
The control of phytopathogenic microorganisms, and in particular, fungi, is of vast economic importance since fungal growth on plants or on parts of plants inhibits production of foliage, fruit or seed, and the overall quality of a cultivated crop. Because of the economic ramifications of fungal propagation in agricultural and horticultural cultivations, a broad spectrum of fungicidal and fungistatic products has been developed for general and specific applications. Fungicides can be separated into two categories according to their fungicidal activity: protectants and curatives. Protectant fungicides, as the name implies, protect the plant against infection. A protectant fungicide must be applied before the pathogen lands on the plant surface and/or the infection process begins. Conversely, a curative fungicide must be able to halt disease development after the infection process has begun. A curative fungicide can be applied after the infection process has begun. Most curative fungicides also have protectant activity.
Inorganic fungicides were generally the first to be used in large-scale crop protection aimed against pathogenic fungi. Notable among these are elemental sulfur applied in powder form, and copper sulfate applied in caustic calcium aqueous mixture. While these inorganic fungicides are generally effective, they have significant drawbacks. The fungicides or derivatives of the fungicides are often environmentally non-recyclable. Additionally, pathogens often develop resistance to synthetic pesticides. Because of the development of resistance, continuous endeavors are needed to develop new crop protecting agents.
A variety of simple structured antimicrobial compounds have been developed. Notable among these are fungicide compositions based on copper, zinc or manganese that have been shown to be effective against a broad range of plant pathogenic fungi and bacteria. Fungicides in this category, unlike the category of inorganic fungicides previously discussed, are generally environmentally friendly and the microbes tend to not develop immunity against them. In certain applications, however, the use of these traditional inorganic fungicides for soil treatment is limited due to the absorption of the metal ions to soil particles.
A need, therefore, remains for antimicrobial compositions that are environmentally safe, cost affordable, and that are highly effective for controlling plant microbes, such as fungi, yeast and bacteria.
RNA interference (RNAi) is a process utilizing endogenous cellular pathways, whereby an interfering RNA (iRNA) molecule (e.g., a dsRNA molecule) that is specific for all, or any portion of adequate size, of a target gene sequence results in the degradation of the mRNA encoded thereby. In recent years, RNAi has been used to perform gene “knockdown” in a number of species and experimental systems; for example, Caenorhabditis elegans, plants, fungi, insect embryos, and cells in tissue culture. See, e.g., Fire et al. (1998) Nature 391:806-811; Martinez et al. (2002) Cell 110:563-574; McManus and Sharp (2002) Nature Rev. Genetics 3:737-747; Koch and Kogel (2014) Plant Biotech. J. 12:821-831.
RNAi accomplishes degradation of mRNA through an endogenous pathway including the DICER protein complex. DICER cleaves long dsRNA molecules into short fragments of approximately 20 nucleotides, termed small interfering RNA (siRNA). The siRNA is unwound into two single-stranded RNAs: the passenger strand and the guide strand. The passenger strand is degraded, and the guide strand is incorporated into the RNA-induced silencing complex (RISC). Micro ribonucleic acid (miRNA) molecules may be similarly incorporated into RISC. Post-transcriptional gene silencing occurs when the guide strand binds specifically to a complementary sequence of an mRNA molecule and induces cleavage by Argonaute, the catalytic component of the RISC complex. This process is known to spread systemically throughout some eukaryotic organisms, despite initially limited concentrations of siRNA and/or miRNA, such as plants, nematodes, and some insects.
Only transcripts complementary to the siRNA and/or miRNA are cleaved and degraded, and thus the knock-down of mRNA expression is sequence-specific. In plants, several functional groups of DICER genes exist. The gene silencing effect of RNAi persists for days and, under experimental conditions, can lead to a decline in abundance of the targeted transcript of 90% or more, with consequent reduction in levels of the corresponding protein. In fungi, there are two DICER enzymes, where DICER2 is the major enzyme participating in post-transcriptional gene silencing. On the other hand, DICER1 has a redundant role in the pathway (Catalanotto. C., et al., (2004) Redundancy of the two dicer genes in transgene-induced posttranscriptional gene silencing in Neurospora crassa. Molecular Cell Biology 24:2536-2545).